Figure 1.
R50E is a dominant-negative inhibitor of FGF signaling.
A.R50E competed with WT FGF1 for binding to the FGFR1 D2D3 fragment. Biotinylated FGF1 and increasing concentrations of unlabelled FGF1 or FGF1 mutants were incubated with the immobilized FGFR1 D2D3 fragment and bound biotinylated FGF1 was measured with HRP-conjugated avidin. The 3xA mutation located at the predicted FGF-FGFR binding site [1] was used as a negative control. The results indicate that R50E competitively blocked the binding of biotinylated WT FGF1 to FGFR fragment to the same extent as WT FGF1. * P<0.0001, ** P = 0.0002 (n = 3) compared to +3A. There is no significant difference between WT and R50E at 10 µg/ml. B. R50E suppressed the DNA synthesis in BaF3-FGFR1c cells induced by WT FGF1. We cultured BaF3-FGFR1c cells in the presence of 1 ng/ml WT FGF1 and 25 or 50 ng/ml R50E for 24 h instead of IL-3 and measured incorporation of BrdU. Results are shown as means +/−SEM. * P<0.0001, ** P = 0.0003 by t-test (n = 4) compared to No R50E. C. R50E suppressed the proliferation of BaF3-FGFR1c cells induced by WT FGF1. We cultured BaF3-FGFR1c cells in the presence of 1 ng/ml WT FGF1 and 100 or 200 ng/ml R50E for 24 h instead of IL-3 and measured cell proliferation by MTS assays. Results are shown as means +/− SEM. * P<0.0025, ** P = 0.0093 by t-test (n = 3) compared to No R50E.
Figure 2.
WT FGF1 induced FGFR1-FGF-αvβ3 ternary complex formation, but R50E was defective in this function.
We immunoprecipitated the FGFR1-αvβ3 complex from cell lysates with anti-FGFR1 (A) or anti-β3 mAb (B), and analyzed the immuno-purified materials by Western blot analysis. A. WT FGF1 induced co-immunoprecipitation of integrin β3 and SHP-1 with FGFR1 using anti-FGFR1, but R50E was defective in this function. B. WT FGF1 induced co-immunoprecipitation of FGFR1 with integrin β3 using anti-integrin β3, but R50E was defective in this function. We stimulated serum-starved NIH 3T3 cells with 5 ng/ml WT FGF1 or R50E for 1 h in the presence of 5 µg/ml heparin. C. Co-precipitation of integrin β3 and FGFR1 upon FGF1 stimulation in HUVEC. Serum-starved HUVEC were stimulated by 5 ng/ml WT FGF1 or R50E with 5 µg/ml heparin for 1 h. Cell lysates were immunoprecipitated with anti-FGFR1 or anti-β3 monoclonal antibody, and the immunoprecipitates were analyzed by Western blotting. D. WT FGF and R50E similarly activated c-Src. We stimulated serum starved NIH 3T3 cells with WT FGF1 or R50E, and cell lysates were analyzed by Western blotting using antibodies specific to phospho-c-Src or c-Src. E. Time-course of embryonic fibroblasts from SHP-2 (−/−) or control mice. Serum starved cells were treated with WT FGF1 or R50E (5 ng/ml) for the time indicated and cell lysates were analyzed by Western blotting.
Figure 3.
R50E is less effective in inducing sustained ERK1/2 activation in NIH3T3 cells.
A. Time-course of ERK1/2 activation and FRS2α activation induced by WT FGF1- or R50E-stimulated cells. We stimulated serum-starved cells with 5 ng/ml WT FGF1 or R50E at indicated time points and analyzed cell lysates by Western blotting using anti phospho-FRS2α, phospho-ERK1/2, total-FRS2α, or ERK1/2 antibody. A representative data is shown from several independent experiments. B. Time-course of WT FGF1 or R50E induced FGFR1 phosphorylation. We stimulated serum starved cells with 5 ng/ml WT FGF1 or R50E in the presence of 5 µg/ml heparin, and analyzed cell lysates by Western blotting. A representative data is shown from several independent experiments. C. ERK1/2 activation levels more rapidly reduced in cells stimulated with R50E than cells stimulated with WT FGF1. Lysates of cells stimulated with R50E or WT FGF1 were analyzed by Western blotting using anti phospho- or total FRS2α antibody. Fold increase of ERK1/2 signals (phosphorylated protein/total protein) is shown with control “time 0” as 1. Data are shown as means +/− SEM of triplicate experiments. ERK1/2 activation at 6 h with R50E is significantly lower in than with WT FGF1 (* P<0.05, n = 3). D. FRS2α phosphorylation levels reduced more rapidly in cells stimulated with R50E than in cells stimulated with WT FGF1. Lysates of cells stimulated with R50E or WT FGF1 were analyzed by Western blotting using anti phospho- or total FRS2α antibody. Relative intensity of FRS2α signals is shown with density at 15 min as 100. The level of total FRS2α in each lane was comparable using anti-FRS2α (data not shown). FRS2α phosphorylation at 6 h with R50E is significantly lower in than with WT FGF1 (* P<0.05, n = 3). E. Time-course of ERK1/2 activation induced by WT FGF1-or R50E-stimulated human umbilical endothelial cells (HUVECs). Experiments were performed as described in A except that HUVEC was used.